AMP selection of HEK293
July 19, 2006 at 12:35 pm #5267szkorneliaParticipant
I am transfecting HEK293 cells, and trying to make a stable cell line containing an Amp resistant plasmid construct. Does anyone can tell me the concentration of the Amp I should use when I am trying to select the plasmid containing cells? Any help would be appreciated.
July 19, 2006 at 7:11 pm #51758TerAleParticipant
The "Amp", if I understood right, is the ampicillin resistance gene. Ampicillin is the prokariotic antibiotic and is used for selection of bacteria. To select transfected eukariotic cells (HEK293 or any other) your plasmid should have a gene providing the resistance to an eukariotic antibiotic, which is in your case, most probably, the Neo (neomicin) resistancy gene. Check your plasmid for gene like this. If there is no genes for eukariotic antibiotic resistance then you have to use another plasmid.
July 19, 2006 at 7:14 pm #51759TerAleParticipant
Sure, it should be "neomycin".
July 20, 2006 at 1:54 am #51764damien jamesParticipant
@TerAle: He probably knows all that. That not telling nothing, just basic micro. He just want concentration of Amp.
You should use multiple Amp dilution instead of just one single one and plate multiple times with different dilution factor. Well, you know. I would start with 250-300 mg per mL. But I would not dilute by 10s because that is too much. Maybe 1/2s or less. To like 150, 100, you get idea.
July 20, 2006 at 1:59 am #51767canalonParticipant
Well most of the plasmid I have used were selected with 100 mg/L or ug/mL, not 200 or 300 mg/ml. That would be expensive too 😉
But the way the question is asked I think that TerAle makes a more interesting point.
July 20, 2006 at 2:21 am #51769damien jamesParticipant
I thought szkornelia was only asking for concentration of Amp, since that was his question. If you are selecting for Amp resistant HEK293 line and do not know already micro, then you should not be messing with it in first place.
Also you are right, it should be ug/mL. I made mistake in post. Do not know what I was thinking. I was thinking about how we buy Amp instead of dilution with plate or broth. Do you use trihydrate form? We buy in several different dilution. 250mg/ml, then other are about half that.
July 20, 2006 at 6:27 am #51779szkorneliaParticipant
Thanks for everybody. I meant Neo, I don’t know why did I write Amp. Maybe because I was picking colonies from the transformed bacterial plate today, and the procaryotic resistancy gene in the construct is Amp. Thanks again for the suggestions.
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